Method of cryopreserving selected sperm cells

Abstract

The present invention provides a method of cryopreserving sperm that have been selected for a specific characteristic. In a preferred embodiment, the method is employed to freeze sex-selected sperm. Although the cryopreservation method of the invention can be used to freeze sperm selected by any number of selection methods, selection using flow cytometry is preferred. The present invention also provides a frozen sperm sample that has been selected for a particular characteristic, such as sex-type. In preferred embodiments, the frozen sperm sample includes mammalian sperm, such as, for example, human, bovine, equine, porcine, ovine, elk, or bison sperm. The frozen selected sperm sample can be used in a variety of applications. In particular, the sample can be thawed and used for fertilization. Accordingly, the invention also includes a method of using the frozen selected sperm sample for artificial insemination or in vitro fertilization.

1. A method of freezing separated sperm cells, comprising the steps of:
 a. obtaining sperm cells from a male of a species of non-human mammal; 
 b. differentiating said sperm cells based upon at least one property which allows a portion of said sperm cells to be identified as bearing either an X-chromosome or a Y-chromosome; 
 c. separating said sperm cells into a discrete population based upon said at least one property; 
 d. establishing from said discrete population a selected sperm sample having a sufficient number of sperm cells to fertilize an egg of a female of said species of non-human mammal; 
 e. freezing said selected sperm sample. 
 
     
     
       2. A method of freezing separated sperm cells as described in  claim 1  wherein said step of differentiating said sperm cells based upon a property which allows a portion of said sperm cells to be identified as either an X chromosome bearing sperm cell or a Y chromosome bearing sperm cell comprises assessing the amount of DNA within each sperm cell. 
     
     
       3. A method of freezing separated sperm cells as described in  claim 2  further comprising the step of staining said sperm cells a fluorochrome which binds to DNA within said sperm cells. 
     
     
       4. A method of freezing separated sperm cells as described in  claim 3  wherein said step of assessing the amount of DNA within each sperm cell comprises the steps of:
 a. exciting said fluorochrome which binds to DNA within said sperm cells; and 
 b. analyzing light emitted by said fluorochrome bound to DNA within said sperm cells. 
 
     
     
       5. A method of freezing separated sperm cells as described in  claim 1  wherein said step of separating said sperm cells having at least one desired sperm cell characteristic into a discrete population, further comprises:
 a. entraining sperm cells within a fluid stream; 
 b. oscillating said fluid stream to generate droplets containing one of said sperm cells; 
 c. charging each of said droplets based upon said at least one property differentiated; and 
 d. deflecting said droplets based upon droplet charge. 
 
     
     
       6. A method of freezing separated sperm cells as described in  claim 1  further comprising the step of removing fluid suspending said discrete population of sperm cells. 
     
     
       7. A method of freezing separated sperm cells as described in  claim 6  further comprising the step of equilibrating said discrete population of sperm cells in a final extender. 
     
     
       8. A method of freezing separated sperm cells as described in  claim 7  wherein said step of equilibrating said discrete population of sperm cells in a final extender has a duration of not less than about 3 hours and not more than about 6 hours. 
     
     
       9. A method of freezing separated sperm cells as described in  claim 7  wherein said final extender comprises egg-yolk-Tris containing about 6% glycerol. 
     
     
       10. A method of freezing separated sperm cells as described in  claim 7  wherein said final extender comprises egg-yolk-TES-Tris containing about 5% glycerol. 
     
     
       11. A method of freezing separated sperm cells as described in  claim 7  wherein said final extender comprises a cold shock treatment, an energy source, an antibiotic, and a cryoprotectant. 
     
     
       12. A method of freezing separated sperm cells as described in  claim 11  wherein said energy source is selected from the group consisting of a saccharide, a glucose, a fructose, a 56 mM fructose, a 45 mM to 60 mM fructose, a mannose, and any combination thereof. 
     
     
       13. A method of freezing separated sperm cells as described in  claim 11  wherein said antibiotic is selected from the group consisting of tylosin, gentamicin, lincomycin, linco-spectin, spectinomycin, penicillin, streptomycin, and any combination thereof. 
     
     
       14. A method of freezing separated sperm cells as described in  claim 11  wherein said cryoprotectant is selected from the group consisting of a disaccharide, a trisaccharide, and any combination thereof. 
     
     
       15. A method of freezing separated sperm cells as described in  claim 11  wherein said cryoprotectant is selected from the group consisting of glycerol, 6% glycerol, between 5% to 7% glycerol, dimethyl sulfoxide, ethylene glycol, propylene glycol, and any combination thereof. 
     
     
       16. A method of freezing separated sperm cells as described in  claim 7  wherein said final extender comprises a mixture of sodium citrate, egg yolk, glycerol, and at least one antibiotic. 
     
     
       17. A method of freezing separated sperm cells as described in  claim 16  wherein said mixture further comprises fructose. 
     
     
       18. A method of freezing separated sperm cells as described in  claim 16  wherein said mixture further comprises milk. 
     
     
       19. A method of freezing separated sperm cells as described in  claim 16  wherein said mixture further comprises Tris [hydroxymehtyl]aminomethane. 
     
     
       20. A method of freezing separated sperm cells as described in  claim 16  wherein said mixture further comprises Tris[hydroxymehtyl]-2-aminoethanesulfonic acid. 
     
     
       21. A method of freezing separated sperm cells as described in  claim 7  wherein said final extender further comprises a component which maintains osmolality and buffers pH. 
     
     
       22. A method of freezing separated sperm cells as described in  claim 21  wherein said component which maintains osmolality and buffers pH is selected from the group consisting of a buffer comprising a salt, a buffer containing a carbohydrate, and any combination thereof. 
     
     
       23. A method of freezing separated sperm cells as described in  claim 21  wherein said component which maintains osmolality and buffers pH is selected from the group consisting of sodiumcitrate, Tris[hydroxymethyl]aminomethane, 200 mM Tris [hydroxymethyl]aminomethane, 175 mM to 225 mM Tris[hydroxymethyl]aminomethane, 200 mM Tris[hydroxymethyl]aminomethane/65 mM citric acid monohydrate, 175 mM to 225 mM Tris[hydroxymethyl]aminomethane/50 mM to 70 mM citric acid monohydrate, N-Tris [hydroxymethyl]methyl-2-aminoethanesulfonic acid, 200 mM Tris[hydroxymethyl]methyl1-2-aminoethanesulfonic acid, 175 mM to 225 mM Tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid, 200 mM Tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid/65 mM citric acid monohydrate, 175 mM to 225 mM Tris[hydroxymethyl]methyl1-2-aminoethanesulfonic acid/50 mM to 70 mM citric acid monohydrate, monosodium glutamate, milk, HEPES buffered medium, and any combination thereof. 
     
     
       24. A method of freezing separated sperm cells as described in  claim 11  wherein said cold shock treatment is selected from the group consisting of egg yolk, 20% egg yolk, 15% to 25% egg yolk, an egg yolk extract, milk, a milk extract, casein, albumin, lecithin, and any combination thereof. 
     
     
       25. A method of freezing separated sperm cells as described in  claim 1  wherein said step of freezing said discrete population of sperm cells having at least one desired sperm cell characteristic comprises establishing an insemination sample having a sufficient number of sperm cells to inseminate a female of said species of mammal and to fertilize at least one egg. 
     
     
       26. A method of freezing separated sperm cells as described in  claim 25  wherein said discrete population of sperm cells having at least one desired sperm cell characteristic comprises sex-selected sperm cells. 
     
     
       27. A method of freezing separated sperm cells as described in  claim 26  wherein said insemination sample contains between about one million and about three million sex-selected sperm cells. 
     
     
       28. A method of freezing separated sperm cells as described in  claim 1  wherein said sperm cells are bovine sperm cells. 
     
     
       29. A method of freezing separated sperm cells as described in  claims 1  wherein said sperm cells are equine sperm cells. 
     
     
       30. A method of freezing separated sperm cells as described in  claim 1  wherein said sperm cells are porcine sperm cells. 
     
     
       31. A frozen separated cell sample in accordance with the method of  claim 1 . 
     
     
       32. A method of freezing separated sperm cells as described in  claim 1  wherein said step of separating said sperm cells having at least one desired sperm cell characteristic into said discrete population comprises the step of separating said sperm cells by a separation method selected from a group consisting of flow cytometry and fluorescence-activated cell sorting. 
     
     
       33. A method of freezing separated sperm cells as described in  claim 1  and further comprising the step of cooling said sperm cells. 
     
     
       34. A method of freezing separated sperm cells as described in  claim 33  wherein said step of cooling said sperm cells comprises the step of reducing the temperature of said sperm cells to about 5° Celsius.